Effects of sera, hormones and granulosa cells added to culture medium for in-vitro maturation, fertilization, cleavage and development of bovine oocytes

Sera (fetal calf serum: FCS; and oestrous cow serum: ECS), hormones (2.5 FSH micrograms/ml + 5 micrograms LH/ml + 1 microgram oestradiol/ml) and granulosa cells (5 x 10(6)/ml) were added to culture medium to determine the frequencies of in-vitro maturation, fertilization, cleavage (2- to 8-cell) and development into blastocysts of bovine follicular oocytes. The maturation rates after 24 h in culture were not significantly different among the three factors tested (56-72%). The fertilization rates were significantly affected by serum type and the addition of granulosa cells. FCS gave significantly higher rates of fertilization (57-71%) than did ECS (34-52%), but the proportions of polyspermic fertilization were significantly higher in the former (8-19%) than in the latter (2-3%). The addition of hormones did not affect fertilization, cleavage and development. Neither type of serum affected cleavage and development. The highest rates of blastocyst formation were obtained when granulosa cells alone were added (FCS, 17%; ECS, 16%). The cell numbers of the blastocysts obtained were 100-150, similar to those of blastocysts developed in vivo. Transfer of 6 blastocysts to 3 cows resulted in 1 pregnancy. The present results indicate that the co-culture with granulosa cells is the most important factor for in-vitro fertilization to development into blastocysts of bovine oocytes matured in vitro.     

Induction of human microvascular endothelial tubular morphogenesis by human keratinocytes: involvement of transforming growth factor-alpha.

Transforming growth factor-alpha(TGF-alpha), homologous to epidermal growth factor(EGF), is closely involved in hyperproliferation of human keratinocytes. Psoriasis is a common hyperproliferative skin disease characterized by hyperproliferation of keratinocytes and abnormal development of dermal capillary networks. In this study, we have examined whether keratinocytes could enhance angiogenesis. TGF-alpha or EGF efficiently stimulated formation of tubular-like structures of human omental microvascular endothelial(HOME) cells in type I collagen gels. Human keratinocytes produced TGF-alpha. To examine whether co-cultured keratinocytes could induce tubulogenesis of HOME cells in collagen gel, we have developed a co-culture system with human keratinocytes. Surprisingly, there appeared new development of many tubular-like structures of HOME cells in collagen gels when co-cultured with keratinocytes. This keratinocytes-dependent tubulogenesis was almost completely blocked when anti-TGF-alpha-antibody was present. The TGF-alpha molecules derived from keratinocytes appeared to enhance tubulogenesis of human microvascular endothelial cells. We propose the hypothesis that secretory TGF-alpha from human keratinocytes may promote an autocrine loop to proliferate the skin keratinocytes and also a paracrine loop to induce the skin angiogenesis.     

Two Types of Apoptotic Cell Death of Rat Central Nervous System-Derived Neuroblastoma B50 and B104 Cells: Apoptosis Induced During Proliferation and After Differentiation

Abstract: We describe here two types of apoptotic cell death observed in the rat CNS-derived neuroblastoma B50 and B104 cells. One type was induced by dibutyryl cyclic AMP (DBcAMP) after differentiation, and the other was induced by treatment of proliferating cells with cycloheximide. When B50 and B104 cells were treated with 1 m M DBcAMP in the presence of 0.5% fetal calf serum, they began to extend neurites within 12 h and differentiated into neurons at 24 h, as reported previously. However, further cultivation with DBcAMP for up to 72 h led to flotation and, finally, death. Death was by apoptosis as shown by chromatin condensation and DNA fragmentation. Addition of a protein kinase A inhibitor or removal of DBcAMP after differentiation suppressed apoptosis, indicating the involvement of cyclic AMP and protein kinase A in apoptotic cell death. Cell death was also induced in proliferating cells without neurite outgrowth by treatment with cycloheximide. The death was also judged to be by apoptosis based on chromatin condensation and apoptotic body formation, although DNA fragmentation into small sizes was not detected. Both types of cell death showed similar responses to inhibitors for protein kinases and protein phosphatases.     

Immunohistochemical characteristics of the constituents of senile plaques and amyloid angiopathy in aged cynomolgus monkeys

Abstract: In this study, we immunohistochemically examined the several constituents of senile plaques (SPs) and cerebral amyloid angiopathy (CAA) in aged cynomolgus monkeys. Apolipoprotein E (apoE) deposited in all mature plaques and CAA, and in half of the diffuse plaques. Alpha-1-antichymotripsin (αACT) deposited in half of the mature plaques and in one third of the CAA. Amyloid precursor protein (APP), ubiquitin (Ub), and microtubule-associated protein-2 (MAP-2) accumulated in the swollen neurites of mature plaques. Glial fibrillary acidic protein (GFAP) was detected in the astrocytes and their processes surrounding the mature plaques. Tau was detected in neither the SPs nor CAA. Therefore, mature plaques involved extracellular Aβ, apoE, and αACT, and also astrocytes and swollen neurites. However, diffuse plaques involved only extracellular Aβ and apoE. Since these features, except for tau, were consistent with those in humans, this animal model will be useful for studying the pathogenesis of cerebral amyloid deposition.     

Plasmid formation and its relation to the formation of spontaneously developing pocks in Streptomyces azureus ATCC 14921

Streptomyces azureus ATCC 14921 harboured a plasmid pSA1 together with its chromosomal integrated sequence (pSA1_int). The att P site on the plasmid was located at ca 170 bp Bam HI- Sph I fragment by site-specific integration. The free form was generated from the integrated sequence during the development of its host mycelia in the solid culture, but not in the liquid culture. The free form seemed to elicit the formation of spontaneously developing pocks on its host mycelia in the solid culture.     

Nucleotide Sequence Surrounding the Locus Marker D21S246 on Human Chromosome 21

Most ofthe human Not I linking clones identified to date areconsidered to be derived from CpG islands because ofthe recognitionsequence of this enzyme, and CpG islands have been reportedto be located around the 5' regions of genes. As a pilot study,we determined the complete nucleotide sequence (41,924 bp) ofa human cosmid clone (LL21NC02Q7A10) containing the marker D21S246originating from a Not I linking clone. As a result of sequenceanalysis, we successfully mapped and revealed the genomic genestructure for KIAA0002 previously reported as a cDNA clone.This gene consists of 15 exons and was shown to exist at theD21S246 locus on human chromosome 21q21.3–q22.1. Theseresults demonstrated that genomic marker-anchored DNA sequencingis a useful approach for the human genome project.     

Identification of a putative RNA helicase (HRH1), a human homolog of yeast Prp22.

In the budding yeast Saccharomyces cerevisiae, a number of PRP genes known to be involved in pre-mRNA processing have been genetically identified and cloned. Three PRP genes (PRP2, PRP16, and PRP22) were shown to encode putative RNA helicases of the family of proteins with DEAH boxes. However, any such splicing factor containing the helicase motifs in vertebrates has not been identified. To identify human homologs of this family, we designed PCR primers corresponding to the highly conserved region of the DEAH box protein family and successfully amplified five cDNA fragments, using HeLa poly(A)+ RNA as a substrate. One fragment, designated HRH1 (human RNA helicase 1), is highly homologous to Prp22, which was previously shown to be involved in the release of spliced mRNAs from the spliceosomes. Expression of HRH1 in a S. cerevisiae prp22 mutant can partially rescue its temperature-sensitive phenotype. These results strongly suggest that HRH1 is a functional human homolog of the yeast Prp22 protein. Interestingly, HRH1 but not Prp22 contains an arginine- and serine-rich domain (RS domain) which is characteristic of some splicing factors, such as members of the SR protein family. We could show that HRH1 can interact in vitro and in the yeast two-hybrid system with members of the SR protein family through its RS domain. We speculate that HRH1 might be targeted to the spliceosome through this interaction.